JP2021188960A - Test strip for immunochromatography - Google Patents

Abstract

To provide a test strip for immunochromatography, with which it is possible to both shorten the detection time and improve detection sensitivity.SOLUTION: Provided is a test strip for immunochromatography, comprising: a sample supply unit to which a sample that could contain a substance to be detected is supplied; a conjugate unit including a conjugate in which an antibody or an antigen immunologically reacting upon the substance to be detected is immobilized to a marker; and a detection unit for supplementing a composite that includes the substance to be detected and the conjugate. The conjugate unit is formed on a nonwoven fabric made of synthetic fiber, the water absorption speed of which is 1 to 20 second/cm.SELECTED DRAWING: Figure 1

Description

The present invention relates to a test strip for immunochromatography and a method for detecting a substance to be detected using the test strip.

As one of the methods for detecting a substance to be detected in a sample by an antigen-antibody reaction, a measurement method using a test strip for later later chromatography has been conventionally performed. In the immunochromatography, an antibody or an antigen against an antigen or an antibody to be detected is immobilized on an insoluble membrane carrier which is a chromatograph medium to create a detection unit which is a stationary phase. Then, a labeled substance sensitized by an antibody or antigen capable of binding to the substance to be detected, which is a conjugate (detection reagent), is used as a mobile phase, and the substance to be detected and the conjugate, which is a mobile phase, are specifically reacted. Let me. Further, in the detection unit which is a stationary phase, the substance to be detected bound to the conjugate is specifically reacted with the antibody or antigen immobilized on the detection unit. Then, since colloidal metal particles such as gold colloid and color latex particles are usually used as the labeled substance, the presence or amount of the substance to be detected in the sample is detected from the color in the detection unit. ..

Various techniques have been publicly disclosed as techniques for test strips for immunochromatography. For example, as a technique for shortening the detection time and improving the detection sensitivity, Patent Document 1 describes a liquid absorption rate that contributes to shortening the inspection time while maintaining a fiber suitable for a lateral flow type in vitro diagnostic agent. An in vitro diagnostic sample pad, which has a high liquid absorption rate and a high release performance of a test solution to a conjugate pad and has a problem of lowering a yield, is disclosed. Further, Patent Document 2 discloses a test strip for lateral flow, which has an object of preventing the accumulation of labeled biomolecules at the interface between the conjugate pad and the membrane.

Japanese Unexamined Patent Publication No. 2012-108031 Japanese Unexamined Patent Publication No. 2012-189355

However, in Patent Document 1, a cellulose-based fiber non-woven fabric is used as a sample pad, and in Patent Document 2, a composite fiber material composed of glass fiber and organic polymer fiber is used as a sample pad, but the detection time is shortened and the detection sensitivity is reduced. There was still room for improvement in achieving a balance between the elution time (development rate) of the conjugate and the reaction time between the conjugate and the substance to be detected.

An object of the present invention is to provide a test strip for immunochromatography capable of achieving both a reduction in detection time and an improvement in detection sensitivity.

Specifically, the present invention relates to the following contents.
<1> A test strip for immunochromatography, which is labeled with a sample supply unit to which a sample that may contain a substance to be detected is supplied, and an antibody or antigen that immunologically reacts with the substance to be detected. The conjugate section includes a conjugate section containing a conjugate immobilized on the body and a detection section that supplements the complex containing the substance to be detected and the conjugate, and the conjugate section has a water absorption rate of 1 to 20 seconds / cm. A test strip for immunochromatography, which is formed on a non-woven material made of synthetic fiber.
<2> The test strip for immunochromatography according to <1>, wherein the non-woven fabric made of synthetic fiber is made of polyester fiber.
<3> The test for later later chromatography according to <1> or <2>, wherein the sample supply unit and the conjugate unit are integrally formed on one synthetic fiber nonwoven fabric at a predetermined distance. strip.
<4> The test strip for laterl flow according to any one of <1> to <3>, wherein the conjugate portion is formed in a line shape orthogonal to the sample development direction.
<5> The immunochromatographic portion according to any one of <1> to <4>, wherein the conjugate portion is formed at a position 1 to 20 mm from the downstream end in the sample development direction in the synthetic fiber nonwoven fabric. Test strip.
<6> When the coloration degree of the detection unit is 100% at the time when twice the reaction completion time elapses, the coloration degree of the detection unit at the reaction completion time is 70% or more. 1> The test strip for immunochromatography according to any one of <5>.

INDUSTRIAL APPLICABILITY According to the present invention, there is provided a test strip for immunochromatography capable of achieving both a reduction in detection time and an improvement in detection sensitivity.

FIG. 1 is a perspective view of a test strip for immunochromatography according to an embodiment. FIG. 2 is a side view of the test strip for lateriometry according to the embodiment. FIG. 3 is a top view of the test strip for lateriometry according to the embodiment. FIG. 4 is a diagram showing the difference in the sample pad portion of the test strip for lateral chromatography.

Hereinafter, the present invention will be described with reference to embodiments, but the present invention is not limited to the following embodiments. In the figure, those having the same function or similar functions are designated by the same or similar reference numerals and the description thereof will be omitted. However, the drawings are schematic. Therefore, the specific dimensions and the like should be determined in light of the following explanations. In addition, it goes without saying that parts having different dimensional relationships and ratios are included between the drawings.

Conventionally, a glass fiber pad has often been used as a sample pad (conjugate portion) of a test strip for immunochromatography. If the glass fiber sample pad is cut when making the test strip, the glass fiber may fall off and adhere to the test strip. If the glass fiber adheres to the detection unit, it may cause an erroneous determination when the device reads the coloration state of the detection unit to make a determination. Therefore, as a material for the sample pad, there has been a demand for a material that does not easily generate dust such as chips at the time of preparation. On the other hand, in order to achieve both a reduction in the detection time and an improvement in the detection sensitivity, it has been required to balance the elution time (development rate) of the conjugate with the reaction time between the conjugate and the substance to be detected. .. However, since it is difficult to achieve both of these problems, a new sample pad that can solve the problems and a test strip for immunochromatography using the same have been required.
According to the present invention, the above-mentioned problems have been solved by using a synthetic fiber pad as a sample pad. As a more preferable embodiment, the above-mentioned problems have been solved by further specifying the position of the conjugate portion. Hereinafter, the present invention will be described through the description of the test strip for immunochromatography according to the embodiment.

[Test strip for immunochromatography]
FIG. 1 is a perspective view of a test strip for immunochromatography according to an embodiment, and FIG. 2 is a side view thereof.
The immunochromatographic test strip 1 according to the embodiment is a sample pad 2 made of synthetic fiber having a sample supply unit 21 to which a sample that may contain a substance to be detected is supplied; and immunology to the substance to be detected. With the conjugate section 23 provided on the sample pad 2 containing the conjugate in which the antibody or antigen that reacts with the present substance is immobilized on the labeled substance; the detection section 31 that captures the complex containing the substance to be detected and the conjugate. , 33 and the antibody-immobilized membrane 3;

(Substances to be detected)
Examples of the substance to be detected include viruses and physiologically active substances such as proteins that can be generally measured by utilizing an antigen-antibody reaction.
Examples of the virus include coronaviruses such as coronavirus 229E, coronavirus OC43, and coronavirus NL63, type A and type B influenza viruses, parainfluenza virus 1, parainfluenza virus 2, and parainfluenza virus 3. Influenza virus, RS virus (respiratory synchronous virus) RS virus such as A type and RS virus B type, adenovirus, rhinovirus, metapneumovirus, cytomegalovirus, bocavirus, hepatitis B virus, hepatitis C virus, Examples of the above-mentioned protein include human hemoglobin, hepatitis B virus antibody, hepatitis C virus antibody, human immunodeficiency virus antibody and the like. Among them, it is preferable to use RS virus and adenovirus as substances to be detected, and it is more preferable to form a plurality of detection parts (for example, two places) described later and use RS virus and adenovirus as substances to be detected.

(sample)
Examples of the sample that may contain the substance to be detected include substances mainly derived from living organisms (organisms) such as body fluids and extracts obtained by extracting the substances to be detected from them. Specific examples of substances derived from living organisms (living organisms) were collected as samples of nasal discharge, nasal discharge, sputum and cotton swabs derived from blood, urine, stool, nostrils, nasal passages, pharynx, nasopharynx, etc. Examples include nasal discharge and saliva. Among them, when the substance to be detected is influenza virus, the samples include nasal discharge and nasal discharge from the nostrils, nasal cavity, pharynx, nasopharynx, and respiratory secretions collected as sputum and cotton swab wiping samples. Is preferable. The above-mentioned substance derived from a living body (living body) or an extract thereof may be used as a sample as it is, or may be appropriately diluted with a diluting solution to form a sample. Further, an appropriately filtered product may be used as a sample.

(Antibody or antigen that immunologically reacts to the substance to be detected)
An antibody or antigen that immunologically reacts to the substance to be detected is an antibody or antigen that can bind to the substance to be detected. Antigens are preferred. An antibody or antigen that immunologically reacts with the substance to be detected is immobilized on a label and a detection unit described later. The antibody or antigen immobilized on the label and the detection unit may be the same, but it is preferable that the label and the detection unit are different. The reason is that in the obtained test strip for later later chromatography, the reaction between the substance to be detected bound to the conjugate and the antibody or antigen of the detection part, and the reaction between the substance to be detected bound to the conjugate and the unreacted conjugate. This is because it is possible to suppress the conflict with. In addition, the reactivity of the substance to be detected bound to the conjugate with the antibody or antigen of the detection unit can be increased, and as a result, the sensitivity of the test strip for later later chromatography becomes good. In addition, another thing means a different kind, an antibody which recognizes a different epitope in the case of an antibody, and an antigen having a different epitope in the case of an antigen.
Further, the antibody immobilized on the label and the detection unit is preferably a monoclonal antibody. By using a monoclonal antibody, the specificity of the reaction can be increased.
Further, in addition to the whole molecule of these antibodies, a functional fragment of an antibody having an antigen-antibody reaction activity is also treated as an antibody in the present invention. Examples of the functional fragment of the antibody include those obtained through an immunization process to an animal, those obtained by using a gene recombination technique, and chimeric antibodies. Examples of the functional fragment of the antibody include F (ab') ₂ , Fab'and the like. These functional fragments can be produced by treating the antibody with a proteolytic enzyme (eg, pepsin, papain, etc.).

(Mark body)
As the labeled body, a known labeled body conventionally used for a test strip for later later chromatography can be used. For example, colloidal metal particles such as gold colloidal particles and platinum colloidal particles, color latex particles, magnetic particles and the like are preferable, and gold colloidal particles are particularly preferable.
As for the particle size of the labeled body, it is preferable to use a labeled body having an appropriate particle size depending on the labeled body to be used. For example, when gold colloidal particles are used as a label, the particle size thereof is preferably 20 to 70 nm, particularly preferably 45 to 65 nm. The above-mentioned colloidal gold particles can be produced by a generally known method, for example, by dropping and stirring a trisodium citrate aqueous solution to a heated tetrachloroauric acid (III) acid aqueous solution.

(Conjugate)
The conjugate is an antibody or antigen that immunologically reacts with the substance to be detected immobilized on the labeled substance as described above. When the substance to be detected is influenza virus, the conjugate is preferably one in which an anti-influenza virus monoclonal antibody is immobilized on gold colloidal particles.
Examples of a method for immobilizing an antibody or antigen that immunologically reacts with a substance to be detected on a labeled substance include physical adsorption, chemical bonding, and the like, and the immobilization is generally performed by physical adsorption. For example, when the anti-influenza virus monoclonal antibody is immobilized on gold colloidal particles, the gold colloidal particles and the anti-influenza virus monoclonal antibody are usually added to a buffer solution and immobilized by physical adsorption.
Further, it is preferable to block the region on the labeled body such as colloidal gold particles to which the antibody or antigen is not bound with BSA or the like.

(Sample supply unit)
The sample supply unit 21 is made of a pad-shaped porous material (sample pad 2) on which a sample can be developed. It is preferable that the sample supplied to the sample supply unit develops a porous material so as to reach the conjugate unit described later.
The sample supply unit 21 is on the upstream side of the sample pad. The sample supply unit 21 is arranged so as to be fluidly connected to the conjugate unit 23 and the detection unit, which will be described later.

The total length of the sample pad, that is, the length from the upstream end to the downstream end of the sample pad can be adjusted to an appropriate length by those skilled in the art, but specifically, 14 to 14 to It can be adjusted to a length of 34 mm, 18-32 mm, or 20-30 mm.

(Conjugate part)
FIG. 3 is a top view of the test strip 1 for immunochromatography according to the embodiment. The conjugate portion 23 is a portion containing the conjugate and may be arranged on the downstream side of the sample supply portion 21, and is downstream of the sample supply portion on the sample pad or independent of other members as a conjugate pad. And may be arranged so as to be fluidly connected to the sample pad and the insoluble membrane carrier upstream of the detection unit. The conjugate portion 23 is preferably formed in a line on the sample pad 2. As shown by the arrow D in FIG. 3, the line-shaped conjugate portion is preferably arranged in a line shape in the direction perpendicular to the sample development direction. In other words, it is preferable that the linear conjugate portion is arranged in a line shape in a direction perpendicular to the longitudinal direction of the immunochromatographic test strip.
It is not necessary that everything on the downstream side of the conjugate portion of the sample pad is a conjugate portion, and a porous material portion that does not contain a conjugate may be present on the further downstream side of the conjugate portion.

As the member forming the conjugate portion, a pad made of non-woven fiber composed of synthetic fiber is preferable. Of these, a pad made of polyester fiber is more preferable.
As the pad made of polyester fiber, for example, a pad having a mass of 50 to 150 g / m ² and a thickness of 0.2 to 0.8 mm per unit area can be used.

As the member forming the conjugate portion, a member having a water absorption rate of 1 to 5 sec / cm is preferable. This is because when the water absorption rate is lower than the above lower limit value, the elution time (development rate) of the conjugate becomes slow and the time until the reaction is completed becomes long. This is because if it is higher than the above upper limit, the antigen / antibody reaction time between the substance to be detected and the conjugate cannot be sufficiently taken, and the reliability of the sensitivity is likely to be impaired.

The position of the conjugate portion can be adjusted to an appropriate length by those skilled in the art. Specifically, as shown by the distance L1 in FIG. 3, the center line (dotted chain line in FIG. 3) in the line width direction of the linear conjugate portion is 1 from the downstream end of the member forming the conjugate portion. It can be arranged on the upstream side by about 30 mm, preferably on the upstream side of 1 to 20 mm, more preferably on the upstream side of 5 to 15 mm, and more preferably on the upstream side of 5 to 9 mm. More preferred. Further, it is preferable that the center line in the line width direction of the conjugate portion is arranged at least 3 mm or more upstream from the upstream end portion of the sample pad. The "center line" of the conjugate portion (detection portion) means the center line drawn in the direction perpendicular to the longitudinal direction of the sample pad as shown by the alternate long and short dash line in FIG. 3, and the longitudinal length of the sample pad. It does not mean a center line drawn in a direction parallel to the direction. The "line width" of the conjugate portion is the length in the longitudinal direction of the sample pad as shown by W in FIG. The line width W of the line-shaped conjugate portion may have a line width sufficient to contain an amount of the conjugate required for detecting the substance to be detected, preferably 0.5 to 15 mm, more preferably 1 to 10 mm. preferable. The line width of the line of the conjugate portion is preferably the same width over the entire line.
It is also conceivable that a part of the line-shaped conjugate portion protrudes to the downstream side and / or the upstream side. In this case, it is sufficient that either the non-protruding portion or the protruding portion has the above-mentioned line width, but it is preferable that the non-protruding portion has the above-mentioned line width.

The conjugate section is arranged so as to fluidly connect the sample supply section on the upstream side in the sample deployment direction and the detection section on the downstream side. For example, when the conjugate portion and the sample supply portion are formed on the same member, the conjugate portion and the sample supply portion are laminated with the insoluble membrane carrier so that the lower surface of the downstream end portion of the member contacts the upper surface of the insoluble membrane carrier described later. May be good.

(Detection unit)
At least one antibody or antigen that immunologically reacts with the substance to be detected is immobilized on the detection unit in a line shape. In the present specification, the insoluble membrane carrier 3 provided with the test line A31 and the test line B33 will be described as an example as the detection unit, but the number of lines of the detection unit is not limited to these and is one. May be more than two. As will be described later, the control line 35 is an arbitrary configuration requirement.
As shown in FIG. 3, the detection unit is preferably arranged in a line in a direction perpendicular to the sample development direction indicated by the arrow D. In other words, the detection unit is preferably arranged in a line in a direction perpendicular to the longitudinal direction of the insoluble membrane carrier.
Immobilization of an antibody or antigen that immunologically reacts with a substance to be detected on an insoluble membrane carrier can be carried out by a conventionally known method. In the case of a lateral flow type test strip for later later chromatography, immobilization is performed as follows. A liquid containing the above antibody or antigen at a predetermined concentration is prepared. Next, the liquid is discharged from the nozzle in the longitudinal direction of the insoluble membrane carrier by using a device having a mechanism capable of moving the liquid in the direction perpendicular to the longitudinal direction of the insoluble membrane carrier while discharging the liquid at a constant speed. It is applied in a line direction perpendicular to the above, for example, in a line width of 1 to 5 mm and 2 to 4 mm, and dried. This makes it possible to form a detection portion on the insoluble membrane carrier. In the present specification, the "line width" of the detection unit is the length of the detection unit in the longitudinal direction of the insoluble membrane carrier. The line width of the line of the detection unit is preferably the same width over the entire line.
It is also conceivable that a part of the line-shaped detection unit protrudes to the downstream side and / or the upstream side. In this case, it is sufficient that either the non-protruding portion or the protruding portion has the above-mentioned line width, but it is preferable that the non-protruding portion has the above-mentioned line width.
When there are a plurality of detectors, the distance between the detectors is not limited and can be adjusted to an appropriate length by those skilled in the art, but specifically, the distance between the center lines of the lines of the detectors. Can be adjusted to a length of about 3 to 6 mm.
The concentration of the antibody or antigen in the above solution is preferably 0.1 to 5 mg / mL, more preferably 0.5 to 2 mg / mL. Further, the amount of the antibody or antigen immobilized on the insoluble membrane carrier can be optimized by adjusting the discharge rate from the nozzle of the above device in the case of the lateral flow type, and is preferably 0.5 to 2 μL / cm. Is.
Further, a liquid containing the above antibody or antigen at a predetermined concentration can be prepared by adding the antibody or antigen to the buffer solution. The buffer solution may further contain salts such as sodium chloride, stabilizers such as sugar, preservatives, preservatives such as proclin, and the like. Salts include those added for the purpose of adjusting the ionic strength, such as sodium chloride, and those added for the purpose of adjusting the pH of the buffer solution, such as sodium hydroxide.
After immobilizing the antibody or antigen on the insoluble membrane carrier, it is also possible to further cover the site other than the site where the antibody or antigen is immobilized by converting a commonly used blocking agent into a solution or vapor to cover the site other than the site where the antibody or antigen is immobilized.
The insoluble membrane carrier may be provided with a control line to ensure the reliability of the assay. At the control line, conjugates that were not captured on the test line or control reagents contained in the sample pad are captured. For example, when the conjugate contains a mouse-derived antibody, an anti-mouse antibody or the like is immobilized, and when the KLH labeled on the sample pad is contained as a control reagent, the anti-KLH antibody or the like is immobilized at the control line position. The position of the control line can be appropriately selected to suit the design of the assay system, but it is desirable to place it on the downstream side of the detection unit.

As the membrane constituting the insoluble membrane carrier, a known membrane that has been conventionally used as an insoluble membrane carrier for a test strip for later later chromatography can be used. Examples thereof include a membrane composed of fibers made of polyethylene, polyethylene terephthalate, nylons, glass, polysaccharides such as cellulose and cellulose derivatives, and ceramics. Specific examples thereof include glass fiber filter paper and nitrocellulose membrane commercially available from Sartorius, Millipore, Toyo Filter Paper, Whatman and the like. The average pore size or reserved particle size of the insoluble membrane carrier is not limited to these, but 0.1 to 20 μm can be used, and 0.5 to 16 μm is more preferable, and 0.7 to 10 μm is more preferable. Is more preferable. The numerical values of these average pore diameters or reserved particle diameters are values measured by ASTM-F316-86.
The total length of the insoluble membrane carrier, that is, the length from the upstream end to the downstream end of the insoluble membrane carrier can be adjusted to an appropriate length by those skilled in the art, but specifically, It can be adjusted to a length of 15-40 mm, 18-35 mm, or 20-30 mm.

(Absorption pad)
In the test strip for later later chromatography, it is preferable to install the absorption pad 5 at the downstream end of the insoluble membrane carrier. The absorption pad is a site having liquid absorbability that controls the development of the sample by absorbing the sample that has moved and passed through the insoluble membrane carrier. As the absorption pad, a known absorption pad conventionally used for a test strip for later later chromatography is used, and for example, filter paper can be used.
The total length of the absorption pad, that is, the length from the upstream end to the downstream end of the absorption pad can be adjusted to an appropriate length by those skilled in the art, but specifically, 20 to 20 to 20. It can be adjusted to a length of 100 mm, 20 to 80 mm, or 20 to 60 mm.

(others)
The test strip for immunochromatography is produced by fixing each member on a solid phase support such as a plastic pressure-sensitive adhesive sheet 6. The solid phase support is composed of a substance that does not interfere with the capillary flow of the sample and conjugate. The adhesive for immobilizing each member on the plastic pressure-sensitive adhesive sheet may be a substance that does not interfere with the capillary flow of the sample and the conjugate due to its components and the like. It is also possible to laminate a polyester film or the like on the test strip for the purpose of increasing the mechanical strength of the insoluble membrane carrier and preventing evaporation (drying) of water during the assay. The immunochromatographic test strip is stored in an appropriate container (housing) considering the size of the immunochromatographic test strip, the sample addition method and addition position, the formation position of the detection part of the insoluble membrane carrier, the signal detection method, and the like. -It can be mounted and used, and the state of being stored and mounted in this way is called a "device".
Further, the test strip for later later chromatography that can be used in the present invention includes a sample supply unit, a conjugate unit and a detection unit, and may further include other reagents and configurations depending on the measurement conditions and the sample. Other reagents include, for example, blocking agents that prevent non-specific reactions, and other configurations include, for example, additional pads for removing components that are not needed for measurement in the sample.

(Manufacturing method of test strip for immunochromatography)
The method for producing the test strip for later later chromatography is not particularly limited, but as a manufacturing procedure, for example, (A) a line-shaped conjugate liquid is applied to a part (a portion to be a conjugate portion) of a pad-shaped porous material. (B) is dried to prepare a sample pad, and (C) is immobilized on a plastic adhesive sheet so that the obtained sample pad and an insoluble membrane carrier having a detection part are in contact with each other for immunochromatography. It is preferably a test strip.

(Method of detecting the substance to be detected using a test strip for later)
The method for detecting the substance to be detected is, for example, the following steps (A) to (D):
(A) The sample supply unit and the substance to be detected are brought into contact with each other;
(B) The substance to be detected in the sample is brought into contact with the conjugate to form a first complex;
(C) The first complex is brought into contact with an antibody or antigen that immunologically reacts with the substance to be detected immobilized on the detection unit to form a second complex; and (D) the detection unit. In the step of confirming the formation of the second complex by measuring the signal derived from the conjugate.

(Other embodiments)
As mentioned above, the invention has been described by embodiment, but the statements and drawings that form part of this disclosure should not be understood to limit the invention. This disclosure will reveal to those skilled in the art various alternative embodiments, examples and operational techniques.
For example, an apparatus including a part of the configuration described in the embodiment can be manufactured in the same manner.
Further, the method described in the examples can be appropriately modified or modified to prepare a test strip for later later chromatography. The signal derived from the conjugate may be measured according to a known method. For example, the signal intensity is semi-quantified by visually comparing the color sample with the signal, or the absorbance or the intensity of the reflected light is measured. do it.
It goes without saying that the present invention includes various embodiments not described here. Therefore, the technical scope of the present invention is defined only by the matters specifying the invention relating to the reasonable claims from the above description.

Next, the present invention will be specifically described with reference to examples, but these do not limit the scope of the present invention.

[Preparation of anti-RS virus monoclonal antibody and anti-adenovirus monoclonal antibody]
The anti-RS virus monoclonal antibody and anti-adenovirus monoclonal antibody used in the following tests were obtained using recombinant influenza nuclear protein as an antigen, immunizing mice, and using the methods normally used by those skilled in the art to produce monoclonal antibodies. Was done.

[Test strip production example 1]
Preparation of test strip for immunochromatography 1) Preparation of gold colloid-labeled RS virus monoclonal antibody (conjugate) 1 Add 1 mL of PBS containing 100 μg / mL anti-RS virus monoclonal antibody to 20 mL of OD / mL colloidal gold solution at room temperature. Was stirred for 10 minutes. Subsequently, 2 mL of a 10% bovine serum albumin (BSA) aqueous solution was added to the colloidal gold solution, and the mixture was stirred for 5 minutes. The resulting solution was centrifuged at 10 ° C. and 10,000 rpm for 45 minutes to remove the supernatant. The residue was suspended in Conjugate Dilution Buffer (manufactured by Scripps) to obtain a conjugate.
2) Preparation of gold colloidal particle-labeled adenovirus monoclonal antibody (conjugate) 1 mL of PBS containing 100 μg / mL anti-adenovirus monoclonal antibody solution was added to 20 mL of gold colloidal solution of OD / mL, and the mixture was stirred at room temperature for 10 minutes. .. Subsequently, 2 mL of a 10% bovine serum albumin (BSA) aqueous solution was added to the colloidal gold solution, and the mixture was stirred for 5 minutes. The resulting solution was centrifuged at 10 ° C. and 10,000 rpm for 45 minutes to remove the supernatant. The sediment was suspended in Conjugate Dilution Buffer (manufactured by Scripps) to obtain a conjugate.

3) Preparation of sample pad As a pad to be a sample pad, a polyester fiber pad having a total length of 28 mm was prepared. The conjugate prepared in 1) above was mixed with 1.33% casein and a 4% sucrose solution (pH 7.5) so as to have a concentration of 8 to 20 OD / mL to prepare a conjugate solution. After that, the conjugate liquid was applied to the prepared polyester fiber pad having a total length of 28 mm, and the distances (L1) from the downstream end of the sample supply section to the center line of the conjugate section were 5 mm and 7 mm, as shown in FIG. , 9 mm, 11 mm, 13 mm, and 15 mm so as to form a line with a line width (W) of 4 mm. It was dried by heating at 70 ° C. for 30 minutes to prepare sample pads 1 to 6 including a conjugate portion. The polyester fiber pad used has a thickness of 0.42 mm and a water absorption rate of 5 seconds / 2 cm.

4) Preparation of insoluble membrane carrier (antibody immobilized membrane) on which anti-RS virus and anti-adenovirus monoclonal antibody is immobilized 1.0 mg / mL anti-RS virus monoclonal antibody, 10 mmol / L phosphate containing 2.5% sucrose Prepare a buffer (pH 7.2) and a 10 mmol / L phosphate buffer (pH 7.2) containing 1.0 mg / mL anti-adenovirus monoclonal antibody and 2.5% sucrose, and apply two types of test lines. It was made into a liquid. A 10 mmol / L phosphate buffer (pH 7.2) containing 1.0 mg / mL anti-mouse IgG monoclonal antibody and 2.5% sucrose was prepared and used as a control line coating solution. A test line coating solution and a control line coating solution were each applied at 1.0 μL / cm on a nitrocellulose membrane using a dispenser for an immunochromatography method “XYZ3050” (manufactured by BIO DOT). The mixture was heated at 70 ° C. for 45 minutes and dried to obtain an antibody-immobilized membrane. At this time, the antibodies were arranged in the order of anti-RS virus monoclonal antibody (test line A; 31), anti-adenovirus monoclonal antibody (test line B; 33), and control antibody (control line; 35) from the upstream end of the membrane. The nitrocellulose membrane used has a thickness of 240 to 280 μm and a flow rate of 90 to 180 s / 40 mm.

4) Preparation of test strip for immunochromatography A test strip for immunochromatography is attached to a plastic adhesive sheet with the antibody-immobilized membrane and absorption pad prepared in 3), respectively, of the sample pads 1 to 6 prepared in 2). 1 to 6 were prepared.

[Test strip production example 2]
As a comparative example, the conjugate portion is provided at 13 mm from the downstream end of the sample pad by the same production method as the test strip 2 for later later chromatography of the test strip production example 1 except that a glass fiber pad having a total length of 28 mm is used. An immunochromatographic test strip 102 with a glass fiber sample pad was made. The glass fiber pad used has a thickness of 0.38 mm and a water absorption rate of 3 seconds / 2 cm.

[Example 1]
The detection test of RS virus and adenovirus was carried out using the test strips 2 and 102 for the later latitus prepared in the above test strip preparation examples 1 and 2.
1. 1. Test method (1) Sample A sample obtained by diluting RS virus antigen and adenovirus antigen 200 to 300 times with PBS (pH 7.4) containing 2% BSA, respectively.
(2) Procedure 120 μL of the sample prepared in (1) above was dropped onto a test strip, and 10 minutes later, the coloration (absorbance) of the test line of the detection unit was measured using an immunochromatographic reader (manufactured by Hamamatsu Photonics).
2. 2. Test Results FIG. 4 shows the absorbance ratio when the sensitivity of the test strip 102 for immunochromatography is 100%. The numerical value is the average value of N = 3.

[Example 2]
The RS virus detection test was carried out using the test strips for later later chromatography prepared in the above test strip preparation examples 1 and 2, and the coloration degree of the test line A was measured.
1. 1. Test method (1) Sample A sample obtained by diluting the RS virus antigen 200-fold with PBS (pH 7.4) containing 2% BSA was used as a sample.
(2) Procedure Drop 120 μL of the sample prepared in (1) above onto the test strip, and adjust the absorbance of the test line A to the immunochromatographic reader (Hamamatsu) 10 minutes after the measurement completion time and 20 minutes after the reaction completion time, which is twice the reaction completion time. It was measured using (manufactured by Photonics).
2. 2. Test results The table shows the absorbance of the test line A at each measurement point of the test strips 1 to 6 and 102 for immunochromatography, and the absorbance ratio at the time of 10 minutes when the absorbance at 20 minutes after the sample was dropped was set to 100%. Shown in 1. All numerical values are average values of N = 3.

From the results shown in FIG. 4, when a polyester fiber pad is used as the sample pad, the detection sensitivity of RS virus is increased by about 10% and the detection sensitivity of adenovirus virus is about 20% as compared with the case of using the glass fiber pad. It was shown to have risen. Conventional glass fiber pads tend to be positively charged, while polyester fiber pads tend to be negatively charged. By using the polyester fiber pad, the release efficiency of the negatively charged conjugate is improved, and it is expected that the sensitivity is increased when the amount of the conjugate involved in the color reaction of the detection part is increased.
From the results in Table 1, the sensitivity ratio of the 10-minute lapse sensitivity to the 20-minute lapse sensitivity ratio of all of the immunochromatographic test strips 1 to 6 is 70% or more, whereas that of the immunochromatographic test strip 102 is 66.7. It was below% and 70%. Therefore, when the sample pad made of polyester fiber is used, the time until the color reaction of the detection part reaches the threshold value is shorter than when the sample pad made of glass fiber is used, and the color reaction is higher in a shorter time. It was shown that chromaticity was obtained. Furthermore, it was also shown that the color reaction proceeds faster in the test strips 1 to 3 in which the distance from the downstream end of the sample pad to the conjugate portion is 9 mm or less.

According to the present invention, it is possible to provide a test strip for immunochromatography in which the time to complete the color reaction is short and the sensitivity is excellent.

1 Test strip for immunochromatography 2 Sample pad 21 Sample supply part 23 Conjugate part 3 Antibody-immobilized membrane 31 Test line A (detection part)
33 Test line B (detector)
35 Control line 5 Absorption pad 6 Plastic adhesive sheet L1 Distance between the downstream end of the sample supply section and the center line of the conjugate section W Line width of the conjugate section

Claims (6)

It is a test strip for immunochromatography,
A sample supply unit to which a sample that may contain a substance to be detected is supplied, and
A conjugate portion containing a conjugate in which an antibody or antigen that immunologically reacts with the substance to be detected is immobilized on a labeled substance, and a conjugate portion thereof.
A detection unit that captures the complex containing the substance to be detected and the conjugate is provided.
The conjugate portion is a test strip for immunochromatography formed on a non-woven fabric made of synthetic fiber having a water absorption rate of 1 to 20 seconds / cm. The immunochromatographic test strip according to claim 1, wherein the synthetic fiber nonwoven fabric is made of polyester fiber. The test strip for later later chromatography according to claim 1 or 2, wherein the sample supply section and the conjugate section are integrally formed on one synthetic fiber non-woven fabric at a predetermined distance. The immunochromatographic test strip according to any one of claims 1 to 3, wherein the conjugate portion is formed in a line shape orthogonal to the sample development direction. The immunochromatographic test strip according to any one of claims 1 to 4, wherein the conjugate portion is formed at a position 1 to 20 mm from the downstream end in the sample development direction of the nonwoven fabric. Claims 1 to 1, when the coloration degree of the detection unit is 100% at the time when twice the reaction completion time elapses, the coloration degree of the detection unit at the reaction completion time is 70% or more. 6. The test strip for immunochromatography according to any one of 6.

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